The purpose of this study was to investigate the physiochemical properties of Doenjang was fermented by added with fungi and protease. The moisture content and pH of Doenjang added with protease (WP) were lower than those of control w/o protease while the contents of titratable acidity, reducing sugar, and amino-type nitrogen in WP were higher than control. The α-amylase activities of Doenjang added with single and mixed Protease B were the highest at 4 weeks of fermentation period and protease activity of WP was about 4 times higher than that of control. The 4-9 kinds of free amino acids (proline, isoleucine, leucine, and phenylalanine etc.) in WP was increased in comparison with control. The DPPH radical scavenging activity and total polyphenol content were higher in WP than control. Total aerobic bacterial and fungal numbers were decreased depending on fermentation time regardless of addition of protease. In conclusion, the protease can be used as additives improving the quality and taste of fermented Doenjang.

Background : The purpose of the present investigation is to enhance extracellular acidic protease production by subjecting a protease producing strain Cordyceps pruinosa DK-01 to random mutagenesis by UV irradiation after ethidium bromide treatment. Methods and Results : Mutants were screened as protease producers on the basis of zone of clearance and relative proteolytic activity (RPA) on skimmed milk agar plates. In addition, mutants showed strong pink-red color intensity and different RAPD profiling compared with wild type control. Four mutants were randomly selected and their extracellular enzyme activities were investigated. In liquid culture without casein, 2.2-, 2.9-, 5.2- and 4.4-fold higher acid protease activity was achieved from mutants DK-m9, -m11 and -m12, respectively, than that of wild type strain (11.13 ± 1.60 U/ml). In liquid culture with casein, 1.1-, 1.3-, 1.3 and 1.3-fold higher acid protease activity was achieved with those mutants were found to produce, respectively, than that of wild type strain (93.95 ± 12.84 U/ml). Maximum acid protease activity was noticed from a mutant DK-m11 in liquid culture with casein (121.18 U/ml) and without casein (57.65 U/ml). The extracellular acid protease produced from DJ-m11 that was active in the pH range 4.5-6.5 and optimum temperature for the activity was 37°C. Furthermore, we found a deformed, shorten structure of setae on the elytron surface of dynastid beetles treated with culture supernatant of the DK-m11. Conclusion : These findings have more impact on enzyme economy for biotechnological and insecticidal applications of fungal proteases.

본 연구에서는 새로운 프로바이오틱스를 선발하고자 protease를 생산하는 GRAS균주를 된장 및 청국장으로부터 분리하고 그 특성을 조사하였다. 분리한 균주를 분리 동정 한 결과 각각B. amyloliquefaciens CDD5, B. amyloliquefaciens CPD4 및B. amyloliquefaciens CGD3로 명명하였다. 분리균 주의 생육은 12시간 배양 시, 7.13~7.32 log CFU/mL로 최대 생육도를 보였으며 24시간까지 유지되다가 감소하는 경향 을 나타내었고, 그 중B. amyloliquefaciens CGD3의 protease 활성이9.21 U/mL로서가장높게나타났다. B. amyloliquefaciens 이 생산하는 protease 활성은 pH 7.0~10.0, 온도는 50℃에서 최적활성을 나타내었으며, casein에서 protease활성이 가장 우수하게 나타났다. pH, NaCl, glucose 농도를 달리한 배양 조건에 따른 B. amyloliquefaciens의 생육특성으로는 pH 5.0~10.0의 넓은 범위에서 생육하였고, NaCl은 12% 농도까 지 증식하였으며 glucose 5%까지는 생육하나 10% 농도부 터는 현저하게 생육이 낮아짐을 확인하였다. 또한 혈당저 하를 유도하는 α-glucosidase의 저해활성을 측정한 결과, B. amyloliquefaciens CPD4 및B. amyloliquefaciens CGD3에 서는 각각 96.65% 및 97.85%의 저해율을 나타내었다. 이러 한 결과는 특히 protease활성이 가장 우수하고 α-glucosidase 또한 높은 저해활성을 가진 B. amyloliquefaciens CGD3 균 주의 생육특성을 고려하여 단백질 분해 분야의 프로바이오 틱 적용 및 당뇨예방용 기능성식품 소재로의 가능성이 기대 된다.

멜라노사이트에서 생성된 멜라노좀은 수상돌기를 따라 케라티노사이트로 이동한다. 세포막을 통한 정보 전달계에 관여하는 protease activated receptor-2 (PAR-2)는 SLIGKV와 같은 펩타이드에 의해 활성화되어 멜라노좀 전달을 증가하는 역할을 한다고 보고되어 있다. 본 연구에서는 새로운 PAR-2의 저해제를 찾고 본 저해제가 멜라노좀의 이동과 색소침착을 저해함을 확인하고자 하였다. PAR-2가 활성화되면 G 단백질이 방출되고, 이때 증가하는 세포 내 칼슘 이온 농도가 lobaric acid에 의하여 감소하는 것을 확인하여 lobaric acid가 PAR-2의 길항제로 작용할 수 있음을 발견하였다. 각질형성세포에서 SLIGKV에 의해 증가된 형광 비드 uptake가 lobaric acid에 의해 억제 되는 것을 확인하였고 또한, 분리된 멜라노좀을 이용한 시험에서도 동일한 경향을 나타내었다. 멜라노사이트와 케라티노사이트를 공동 배양하여 멜라노좀의 이동을 공초점 현미경으로 관찰한 결과, lobaric acid에 의해 멜라노좀의 전달이 억제되었다. 인공피부조직에 lobaric acid를 처리하였을 때 색소 침착이 억제됨을 확인하였고, 또한 Fontana-Masson 염색을 통해 멜라닌의 양이 감소함을 확인하였다. 이상의 결과를 통해 lobaric acid가 PAR-2 길항제로 작용함으로써 케라티노사이트로 멜라노좀 전달을 억제하고 이를 통해 피부 색소 침착을 저해함을 확인할 수 있었다.

Cysteine protease (CP) is one of the well-studied proteolytic enzymes in plants. This class of protease has been implicated in various physiological aspects of developmental stages in plants including seed germination, senescence, and disease immunity. A handful of studies assigned plants cysteine protease in different molecular battlefield under a few selected pathosystems, and initially extricated complex molecular mechanism of resistance. However, its potential use as an agent of resistance to diseases in rice has never been explored. This study demonstrates the function of CP specifically in rice - Xanthomonas oryzae pv. oryzae (Xoo) pathosystem. The CP -encoding full-length cDNA was cloned from Brassica rapa and transformed into japonica rice cv. ‘Gopumbyeo’. The gene was overexpressed under the control of CaMV35S promoter in pFLC vector. Blast analysis of the conserved domain of the gene confirmed its affinity to Peptidase_CIA family. RT-PCR analysis showed that the gene was constitutively expressed in all tissues tested. Regulation of rice resistance through cysteine protease activity is evident in overexpression lines which exhibited an enhanced resistance to four Korean Xoo isolates. Further analyses will be carried out to uncover the specific role of CP in rice-Xoo interaction.

본 연구에서는 protease를 함유하고 있는 파인애플의 산 업적 이용 증대 및 기능성식품 소재 개발을 목적으로 분무 건조공정을 이용하여 파인애플 착즙액을 미세캡슐화 하였 으며 미세캡슐 분말의 물리화학적 특성 및 protease 활성을 조사하였다. 파인애플 착즙액의 pH, 당도 및 protease 활성 은 각각 pH 5.43, 12.80 및 4.82 unit/mL이었다. Protease 활성에 대한 최적 pH 및 온도는 각각 pH 7.0 및 50℃에서 가장 높게 분석되었다. 파인애플 착즙액의 미세캡슐분말 제조는 말토덱스트린 및 알긴산을 피복물질로 사용하여 분무건조하였으며, 수분함량은 3.02~3.75%였다. 색도는 분 무건조 미세캡슐 분말이 동결건조 분말에 비하여 L값 및 a값은 낮고 b값은 높은 경향을 나타내었는데 특히 말토덱스 트린에 알긴산 3% 첨가시 선명한 노란색을 보여주었다. 입자크기는 동결건조 분말(501.57 μm)에 비하여 분무건조 미세캡슐 분말이 42.58~53.32 μm로 유의적으로 작고 균일 한 크기였으며, 입자모양은 전반적으로 구형의 형태를 보 여주어 분말 흐름성이 양호할 것으로 판단되었다. 수분흡 수지수는 말토덱스트린에 알긴산을 3% 첨가한 분무건조 미세캡슐 분말에서 0.41로 가장 낮은 지수를 나타내었으며 수분용해지수는 분무건조 미세캡슐 분말에서 98.22~ 99.76%로 나타나 동결건조 분말보다 우수하였다. 미세캡 슐 분말의 protease 활성은 동결건조 분말(1,297.47 unit/g)이 분무건조 미세캡슐 분말(633.51~692.08 unit/g)보다 유의적 으로 높은 활성을 나타내었으나, in vitro 인체 내 소화모델 에 대한 protease 활성의 안정성은 분무건조 미세캡슐 분말 에서만 g당 23.70~100.83 unit의 효소 활성이 나타나 위액과 장액의 pH 환경에서 안정성을 나타내는 것으로 확인되었 다. 따라서 피복물질로 말토덱스트린 및 알긴산을 첨가하 여 분무건조시 식품산업 활용 측면에서 가공적성이 향상된 미세캡슐 분말의 제조가 가능하고in vitro 인체 내 소화모델 에 대한 protease 활성의 안정성이 우수하여 기능성 식품 소재 개발에 있어 산업적으로 적용 가능할 것으로 사료된 다.

In spite of the overwhelming number of cysteine proteases in plants, only a few were substantially investigated. Papain-like cysteine proteases (PLCPs) are commonly implicated to disease immunity in some key pathosystems in plants, such as in tomato – Cladosporium fulvum, potato/tomato – phytopthora infestans, and Arabidopsis – Ralstonia solanacearum, among the few others. This study demonstrates the function of cysteine protease gene cloned form Brassica rapa (BrCP) related to resistance to Xanthomonas oryzae pv. oryzae in transgenic rice lines. The cysteine protease-encoding full-length cDNA was identified and characterized using web-based tools. The gene is 2,267 bp in size with an open reading frame of 1,365 bp that encodes predicted polypeptide of 455 amino acids. Blast analysis of the conserved domain of the gene confirmed its affinity to Peptidase_CIA family. Full-length cDNA of PLCP in Brassica rapa was then cloned and co-overexpressed in rice with HPT marker. Introgression of the gene was confirmed in the transformants through genomic PCR assay. RT-PCR analysis showed that the gene was constitutively expressed and present in all tissues. The overexpression rice lines exhibited an enhanced resistance when screened with four Korean Xoo isolates.

Bacterial blight is a serious problem of rice in irrigated and rainfed lowlands. It is caused by Xanthomonas oryzae pv. oryzae (Xoo) which is represented by many pathotypes, making it difficult to control. Plant proteases are important players in immunity acting either in the execution of attack, in signaling cascade or in perception of invader. This study demonstrates the response of cysteine protease (CP) upon interaction with the pathogen. The cysteine protease encoding full-length cDNA was identified and characterized using web-based tools. Conserved domain of the gene revealed its affinity to Peptidase_CIA family. The full-length cDNA of CP in Brassica rapa was then cloned and overexpressed in rice. Insertion of gene was verified in the transformants through PCR assay. Spatiotemporal expression of the gene was performed in transgenic rice. To evaluate the resistance of CP-overexpression lines to Xoo, transgenic plants were inoculated with two races of Xoo. In planta analysis of enzymatic activity of CP was also performed before and after infection by the pathogen.

The study was performed to evaluate the effects of soybean meal (T1) and corn distillers dried grains with solubles (DDGS, T2) hydrolyzed by microbial proteolytic enzyme of Protame® on milk production and protein contents in dairy cows. Total of nine Holstein cows (i.e., 1.67 ± 0.47 average parity and 23.7 ± 1.2 kg/d average milk yield) were randomly assigned to control, T1, and T2 (e.g., 3 animals per group) and treated for 4 weeks. Milk yield of 3 different groups was similar in the beginning of the study, however the milk yield of T1 and T2 treatment group had increased by 0.93 kg/d and 1.89 kg/d, respectively. Milk protein level of T2 treatment group was increased by 0.19% (e.g., 0.14 kg/d protein content), whereas there was no significant different in control and T1 group. In conclusion, blood metabolites were ranged in normal level, but BUN content was reduced from 19.03 mg/dl to 17.70 mg/dl in T2 treatment group. This result suggests that DDGS hydrolysate supplement efficiently increase milk yield and milk protein level as well as feed protein availability in dairy cattle.

능소화 추출물의 HIV-1에 대한 항바이러스 효과를 복제 관련 효소에 대한 억제실험과 바이러스 복제억제 실험을 통하여 살펴보았다. 역전사효소 억제활성을 ELOSA 방법으로 실험한 결과 능소화 줄기의 물 추출물이 100 μg/ml 농도에서 각각 37.9%의 활성을 나타내었고, 능소화 잎과 줄기의 메탄올 추출물에서 33.6% 및 31.5%의 HIV-1 protease 억제활성 나타 내었다. 그리고 HIV-1 복제 억제활성은 MT-4 세포에 대한 HIV-1 유도 세포변성억제를 광학현미경으로 관찰하여 살펴본 결과 줄기의 물 추출물이 100 μg/ml 농도에서 HIV-1 바이러스 증식을 완전히 억제하였다.

The tremendous changes of uterine endometrium are observed during early pregnancy and protease and their inhibitors are involved in regulation of cell proliferation and remodeling of the tissues through remodeling the extracellular matrix (ECM). Some of the proteases and protease inhibitors have been suspected to a factor in endometrial changes but many parts of their expression profiles and the physiological roles are not uncovered. To evaluate the functional roles of them, in this study the expression profiles of proteases and protease inhibitors were analyzed using real-time quantitative PCR analysis. Mmp9 (matrix metalloproteinase 9) mRNA levels peaked on day 4 at the time of implantation. On the other hand, Ela2 (neutrophil elastase, NE) mRNA levels were peaked on day 2 of pregnancy. Its expression were decreased until day 4 of pregnancy but increased rapidly until day 7 of pregnancy and decreased again. NE inhibitor Slpi (secretory leukocyte protease inhibitor, SLPI) mRNA levels were related with the implantation stage and with the levels of Ela2. At the time of implantation the expression levels of Slpi mRNA were about 5 times higher than the Ela2 mRNA in the uterus. In the implantation stage embryos, Mmp9 specific mRNA was only detected at the blastocyst. On the other hand, the expression level of SLPI was higher than that of the Ela2 mRNA at blastocyst and 4.5 day p.c. embryos. Based on these results it is suggested that MMP9, SLPI, and NE have important physiological role in embryo implantation both in uterus and embryos.

우리나라 전통발효식품의 하나인 청국장으로부터 고 비활성 세포외 protease 생산능이 우수한 균주를 분리하고 그 특성을 조사하였다. 분리된 균주 중 D14 균주 배양 상징액의 protease 활성이 15.2 U/mL로서 가장 강하였으며 비활성 또한 40.0 U/mg protein으로서 가장 강하게 나타났다. 이 균주의 특성을 조사한 후 Berge's Manual of Systematic Bacteriology에 준하여 Bacillus subti

된장의 영양성과 기능성을 향상시키기 위해 해양심층수와 protease 고 생산성 미생물을 이용하여 개량식 된장을 제조하여 다양한 기능적 특성을 조사하였다. Protease 활성은 재래식 된장에서 분리한 DH3을 접종시킨 된장이 (unit/mL/min)로 가장 우수하였다. Protease 활성과 ACE저해 활성은 발효기간이 길어질수록 증가되었으며 발효 30일 후 된장M과 된장 PD의 동결건조 분말(10mg/mL)의 ACE 저해활성은 각각 , 로 조

Feathers are produced in huge quantities as a waste product at commercial poultry processing plants. Since feathers are almost pure keratin protein, feather wastes represent an alternative to more expensive dietary ingredients for animal feedstuffs. Generally they become feather meal used as animal feed after undergoing physical and chemical treatments. These processes require significant energy and also cause environmental pollutions. Therefore, biodegradation of feather by microorganisms represents an alternative method to prevent environment contamination. The aim of this study was to investigate cultural conditions affecting keratinolytic protease production by Bacillus pumilus RS7. We also assessed the nutritive value of microbial and alkaline feather hydrolysates. The composition of optimal medium for the keratinolytic protease was fructose 0.05%, yeast extract 0.3%, NaCl 0.05%, K2HPO4 0.03%, KH2PO4 0.04% and MgCl2ㆍ6H2O 0.01%, respectively. The optimal temperature and initial pH was 30℃ and 9.0, respectively. The keratinolytic protease production under optimal condition reached a maximum after 18 h of cultivation. Total amino acid content of feather hydrolysates treated by NaOH and B. pumilius RS7 was 113.8 ㎍/ml and 504.9 ㎍/ml, respectively. Essential amino acid content of feather hydrolysates treated by NaOH and B. pumilius RS7 was 47.2 ㎍/ml and 334.0 ㎍/ml, respectively. Thus, feather hydrolysates have the potential for utilization as an ingredient in animal feed.

단백 효소 가수분해물의 천연 항균제로서의 응용성을 검토하기 위하여 casein에 5종의 단백질 가수분해 효소를 작용시켜 얻어진 가수 분해물의 항균활성을 측정하고 활성이 가장 높은 가수분해물을 부분 정제하여 그 응용성을 검토하였다. Casein에 5종 단백질 분해효소를 작용시켜 얻은 가수분해물의 항균활성은 Aspergillus oryzae protease에 의한 것이 가장 높았다. 효소처리에 의하여 얻어진 가수분해 물을 30,000, 10,000,

콩 가수분해물의 기능성 강화를 위하여 콩분말 및 SPI에 4종의 protease를 처리하여 가수분해물의 특성을 조사하였다. 수율은 protease(B)를 처리하였을 때 콩분말에서 43.2%, SPI에서 61.6%로 가장 높게 나타났다. 용해도와 총페놀성 물질은 protease(B)와 (C)처리구에서 크게 증가하였으며, 칼슘내인성은 protease(B)를 처리하였을 때 향상되었다. 콩분말에서 콩 특유의 비린내는 protease처리로 감소하였으나 콩

-2로 동결된 노르웨이산 고등어로부터 pretense 활성이 강한 미생물을 분리하였다. 분리된 미생물을 형태 및 생리학적 성상에 의거하여 동정한 결과 Bacillus magaterium으로 분류되었다. protease 최대 활성을 얻기 위해 최적온도, pH, 배양시간, 탄소원, 질소원, 무기원 및 이들에 대한 지적농도를 조사한 결과 pH 는 8, 온도는 35, 배양시간은 48시간 이였고, 탄소원은 galactose로 1.2%, 질소원은 NHNO

Korean medicinal plants were screened for their inhibitory activity against HIV-1 protease. The inhibitory activity of protease was determined by incubating the extracts in reaction mixtures containing protease and substrate His-Lys-Ala-Arg-Val-Leu-(p-NO2-Phe)-Glu-Ala-Nle-Ser-NH2 to perform proteolytic cleavage reactions. In this study the twenty six extracts from medicinal plants were investigated. Of the extracts tested, the extracts from the stem of Morus alba. exhibited the strongest activity with inhibition of 81% at a concentration of 100μg/ml. The extracts of the flower of Saxjfraga stolonifera, and stems of Euonymus japonica and Castanea crenata showed appreciable inhibitory activity (〉50%) against HIV-1 protease at same concentration.

Bacillus subtilis PANH765 균주가 생산하는 pretense를 황산암모늄에 의한 염석, DEAE-cellulose ion exchange chromatography, Sephacryl S 200 HR 및 Sepharose CL-6B gel filtration을 이용하여 정제하였다. DEAE-cellulose ion exchange chromatography를 행한 결과, 흡착 단백질 부분에서 활성이 높은 분획을 얻었다. 이 분획